SAGE and antibody array analysis of melanoma-infiltrated lymph nodes: identification of Ubc9 as an important molecule in advanced-stage melanomas.

Although patients diagnosed with melanoma of

Biologic therapy.

Biologic therapy has evolved as a fourth treatment option for patients with melanoma and other malignancies. Interrelated advances in molecular diagnostics and gene therapy techniques will further accelerate therapeutic advances in this new arena. A variety of biologic therapeutic options available to the patient with high risk melanoma are reviewed.

Analysis of therapeutic and immunologic effects of R(24) anti-GD3 monoclonal antibody in 37 patients with metastatic melanoma.

BACKGROUND: Antitumor effects of antibodies against ganglioside antigens of melanoma have been reported, but neither optimal doses nor mechanisms have been established. METHODS: This Phase IB trial of the murine immunoglobulin IgG(3) monoclonal antibody R(24) against disialoganglioside GD3 was conducted with 37 patients to define better the dose-response relation and mechanism of action of R(24) in patients with metastatic melanoma. RESULTS: Dose-limiting toxicity consisted of a pulmonary capillary leak syndrome in 3 of 5 patients in the 80 mg/M(2)/day dosage tier. Serial blood and tumor biopsy samples were obtained prior to therapy and on Days 5, 9, and 22 following R(24) infusion. Tumor biopsy-infiltrating lymphocytes were enumerated in peritumoral, endotumoral, and perivascular compartments: endotumoral CD4(+) and CD8(+) T cells and HLA-DR(+) T cells increased over time on R(24) antibody. Endotumoral CD4 lymphoid infiltrate activation (DR expression) and antibody-dependent cytotoxicity were the greatest in the one patient who achieved a complete response. CONCLUSIONS: Clinical response was associated with depression in natural killer (CD56(+) and CD56(+)DR(+)) blood cells (P = 0.03) and was associated with R(24) dosage (P = 0.01). A complete response that lasted 2 years and a partial response that lasted 2 months occurred at a dose of 1 mg/M(2)/day. The limited number of clinical responses observed in this trial hampered the correlation of antitumor and immune parameters but provided a rational foundation for the future evaluation of antiganglioside antibodies.

The separation of anatomic components technique for the reconstruction of massive midline abdominal wall defects: anatomy, surgical technique, applications, and limitations revisited.

Reconstruction of massive abdominal wall defects has long been a vexing clinical problem. A landmark development for the autogenous tissue reconstruction of these difficult wounds was the introduction of "components of anatomic separation" technique by Ramirez et al. This method uses bilateral, innervated, bipedicle, rectus abdominis-transversus abdominis-internal oblique muscle flap complexes transposed medially to reconstruct the central abdominal wall. Enamored with this concept, this institution sought to define the limitations and complications and to quantify functional outcome with the use of this technique. During a 4-year period (July of 1991 to 1995), 22 patients underwent reconstruction of massive midline abdominal wounds. The defects varied in size from 6 to 14 cm in width and from 10 to 24 cm in height. Causes included removal of infected synthetic mesh material (n = 7), recurrent hernia (n = 4), removal of split-thickness skin graft and dense abdominal wall cicatrix (n = 4), parastomal hernia (n = 2), primary incisional hernia (n = 2), trauma/enteric sepsis (n = 2), and tumor resection (abdominal wall desmoid tumor involving the right rectus abdominis muscle) (n = 1). Twenty patients were treated with mobilization of both rectus abdominis muscles, and in two patients one muscle complex was used. The plane of "separation" was the interface between the external and internal oblique muscles. A quantitative dynamic assessment of the abdominal wall was performed in two patients by using a Cybex TEF machine, with analysis of truncal flexion strength being undertaken preoperatively and at 6 months after surgery. Patients achieved wound healing in all cases with one operation. Minor complications included superficial infection in two patients and a wound seroma in one. One patient developed a recurrent incisional hernia 8 months postoperatively. There was one postoperative death caused by multisystem organ failure. One patient required the addition of synthetic mesh to achieve abdominal closure. This case involved a thin patient whose defect exceeded 16 cm in width. There has been no clinically apparent muscle weakness in the abdomen over that present preoperatively. Analysis of preoperative and postoperative truncal force generation revealed a 40 percent increase in strength in the two patients tested on a Cybex machine. Reoperation was possible through the reconstructed abdominal wall in two patients without untoward sequela. This operation is an effective method for autogenous reconstruction of massive midline abdominal wall defects. It can be used either as a primary mode of defect closure or to treat the complications of trauma, surgery, or various diseases.

Gene therapy and tissue engineering.

Gene therapy, the science of altering cellular function or structure at a molecular level, is currently one of the most exciting fields of research across medical specialties. By replacing lost or defective genes or adding genes known to produce proteins with beneficial effects, investigators hope to treat and possibly cure everything from inborn errors of metabolism to acquired diseases, such as emphysema and atherosclerosis. Genetic modification of engineered tissue, the offspring of these two fields, offers the promise of not simply replacing tissue, but improving on the restoration. The studies presented in this article demonstrate many of the principles that form the basis of gene therapy and provide insight into the potential of this emerging technology.

Impaired wound healing and angiogenesis in eNOS-deficient mice.

A role for nitric oxide (NO) in wound healing has been proposed; however, the absolute requirement of NO for wound healing in vivo and the contribution of endothelial NO synthase (eNOS) have not been determined. Experiments were carried out using eNOS gene knockout (KO) mice to determine the requirement for eNOS on wound closure and wound strength. Excisional wound closure was significantly delayed in the eNOS KO mice (29.4 +/- 2.2 days) compared with wild-type (WT) controls (20.2 +/- 0.4 days). At 10 days, incisional wound tensile strength demonstrated a 38% reduction in the eNOS KO mice. Because effective wound repair requires growth factor-stimulated angiogenesis, in vitro and in vivo angiogenesis assays were performed in the mice to assess the effects of eNOS deficiency on angiogenesis. Endothelial cell sprouting assays confirmed in vitro that eNOS is required for proper endothelial cell migration, proliferation, and differentiation. Aortic segments harvested from eNOS KO mice cultured with Matrigel demonstrated a significant reduction in endothelial cell sprouting and [(3)H]thymidine incorporation compared with WT mice at 5 days. Capillary ingrowth into subcutaneously implanted Matrigel plugs was significantly reduced in eNOS KO mice (2.67 +/- 0.33 vessels/plug) compared with WT mice (10.17 +/- 0.79 vessels/plug). These results clearly show that eNOS plays a significant role in facilitating wound repair and growth factor-stimulated angiogenesis.

Systemic interferon-alpha (IFN-alpha) treatment leads to Stat3 inactivation in melanoma precursor lesions.

BACKGROUND: In the setting of familial melanoma, the presence of atypical nevi, which are the precursors of melanoma, is associated with a nearly 100% risk of developing primary melanoma by age 70. In patients with sporadic melanoma, it is estimated that 40-60% of melanomas develop in contiguous association with atypical nevi. Currently, the only way to prevent atypical nevi from progressing to melanoma is to monitor and excise them as soon as they exhibit changes in their clinical features. Activation of the transcription factor, Stat3, has been linked to abnormal cell growth and transformation as well as to interferon alpha (IFN-alpha)-mediated growth suppression in vitro. MATERIALS AND METHODS: To determine whether IFN-alpha, used for adjuvant therapy of high-risk, resected melanoma, induces changes in Stat3 in atypical nevi, patients with a clinical history of melanoma who have multiple atypical nevi were treated for 3 months with low-dose IFN-alpha. Thereupon, the new technology of microscopic spectral imaging and biochemical assays such as electrophoretic mobility shift assays (EMSAs) and immunoblot analysis were used for the study of atypical nevi, obtained before and after IFN-alpha treatment. RESULTS: The results of the investigations provided evidence that, as a result of systemic IFN-alpha treatment, Stat1 and Stat3, which are constitutively activated in melanoma precursor lesions, lose their ability to bind DNA, and as shown in the case of Stat3, become dephosphorylated. CONCLUSIONS: Unlike primary and metastatic melanomas, melanoma precursor lesions cannot be established as cell cultures. Thus, the only way to explore pathways and treatment regimens that might help prevent progression to melanoma is within the context of a melanoma precursor lesion study conducted prospectively. The findings presented here suggest that down-regulation of the transcription factors Stat1 and Stat3 by systemic IFN-alpha treatment may represent a potential pathway to prevent the activation of gene(s) whose expression may be required for atypical nevus cells to progress to melanoma.

Macroscopic spectral imaging and gene expression analysis of the early stages of melanoma.

BACKGROUND: The stages of melanocytic progression are defined as atypical (dysplastic) nevus, melanoma in situ, melanoma in the radial growth phase (RGP), melanoma in the vertical growth phase (VGP), and melanoma in the metastatic growth phase (MGP). Melanoma in situ and RGP melanoma often develop in contiguous association with atypical nevi. This frequently poses a problem with respect to their early detection. Furthermore, unlike cells obtained from VGP and MGP melanomas, cells derived from melanoma in situ and RGP melanoma do not proliferate in vitro. Thus, compared to the late stages of the disease, less information is available regarding genes expressed in the early stages. MATERIALS AND METHODS: To determine whether spectral imaging, a recently developed optical imaging technique, can detect melanoma in situ and RGP melanoma arising in melanoma precursor lesions, atypical nevi in patients with a clinical history of melanoma were subjected to noninvasive macroscopic spectral imaging. To determine at what stage in the progression pathway of melanoma genes having important biological functions in VGP and MGP melanomas are activated and expressed, lesions of melanoma in situ were analyzed by immunohistochemistry and in situ hybridization for expression of some of these known molecular and immunologic markers. RESULTS: The present study demonstrates the capability of noninvasive spectral imaging to detect melanoma in situ and RGP melanoma that arise in contiguous association with atypical nevi. Furthermore, the study provides evidence that genes and antigens expressed in VGP and MGP melanoma are also expressed in melanoma in situ. CONCLUSIONS: Because of the dark and variegated pigmentation of atypical nevi, melanoma in situ and RGP melanoma that arise in these melanoma precursor lesions are often difficult to recognize and thus frequently go unnoticed. The application of new optical screening techniques for early detection of melanoma and the identification of genes expressed in the early stages of melanoma development are two important avenues in the pursuit of melanoma prevention. The investigations presented here document that macroscopic spectral imaging has the potential to detect melanoma in its early stage of development and that genes essential for the proliferation and cell adhesion of VGP and MGP melanoma are already expressed in melanoma in situ.

The effects of soft-tissue expansion on lip repair and midfacial growth in a rabbit cleft lip model.

Midfacial hypoplasia following primary cleft lip repair is a common clinical correlate. Recent experimental work has suggested that increased lip pressure following an undermined lip repair may contribute to midfacial growth inhibition. Soft-tissue expansion has been used to generate additional soft tissue for reconstruction in the cranial region. The use of this technique in the labial region may allow lip repair to be performed with less tension (pressure) and thus facilitate midfacial growth. To test this hypothesis, 40 4-week-old rabbits were randomly divided into four groups. Two groups had surgically created lip and alveolar defects. Ipsilateral labial tissue expanders were placed in all four groups. One cleft and one normal group underwent expansion. The other two groups served as a control. The expanders were removed at 4 weeks, and an undermined lip repair was performed in both cleft groups. Findings revealed that soft-tissue expansion increased labial surface area significantly by approximately 96% (p < 0.001). Labial soft-tissue expansion alone had no effect on midfacial growth during the observation period. In contrast, cleft animals undergoing tissue expansion exhibited significantly reduced (p < 0.05) postoperative lip pressure and increased midfacial growth compared with cleft animals without expansion through 36 weeks of age. Results suggest that preoperative tissue expansion reduced postoperative lip pressure and improved midfacial growth in a rabbit cleft lip model.

Lymphatic mapping with isosulfan blue dye in squamous cell carcinoma of the head and neck.

OBJECTIVE: To determine whether intraoperative lymphatic mapping with isosulfan blue dye and sentinel lymph node biopsy accurately demonstrates the pathway of regional metastases from mucosal sites in squamous cell carcinoma of the head and neck. DESIGN: A prospective clinical study of intraoperative lymphatic mapping. SETTING: An academic tertiary referral center. PATIENTS: Patients with previously untreated squamous cell carcinoma of the head and neck whose surgical treatment included neck dissection. INTERVENTION: Injection of isosulfan blue dye into the mucosa surrounding squamous cell carcinomas of the upper aerodigestive tract during cervical lymphadenectomy. OUTCOME MEASURES: Correlation of the pathologic findings in the blue sentinel lymph node with those in the remaining cervical lymphatics. RESULTS: No blue-stained cervical lymphatics were identified after injection of the mucosa surrounding the primary squamous cell carcinoma with isosulfan dye. CONCLUSION: The technique of intraoperative lymphatic mapping with isosulfan blue dye requires further study before it can be used for the detection of occult cervical metastases in squamous cell carcinoma of the head and neck.

Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer.

Most evidence indicates that nitric oxide plays a role in normal wound repair; however, involvement of inducible nitric oxide synthase (iNOS) has not been established. Experiments were carried out to determine the requirement for iNOS in closing excisional wounds. Wound closure was delayed by 31% in iNOS knockout mice compared with wild-type animals. An identical delay in wound closure was observed in wild-type mice given a continuous infusion of the partially selective iNOS inhibitor N6-(iminoethyl)-L-lysine. Delayed wound healing in iNOS-deficient mice was completely reversed by a single application of an adenoviral vector containing human iNOS cDNA (AdiNOS) at the time of wounding. Reverse transcription PCR identified iNOS mRNA expression in wild-type mice peaking 4-6 d after wounding, and confirmed expression of human iNOS in the adenoviral vector containing human iNOS cDNA-treated animals. These results establish the key role of iNOS in wound closure, and suggest a gene therapy strategy to improve wound healing in iNOS-deficient states such as diabetes, and during steroid treatment.

Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies.

A Phase I dose escalation trial of i.v. administered recombinant human interleukin 12 (rhIL-12) was performed to determine its toxicity, maximum tolerated dose (MTD), pharmacokinetics, and biological and potential antineoplastic effects. Cohorts of four to six patients with advanced cancer, Karnofsky performance >/=70%, and normal organ function received escalating doses (3-1000 ng/kg/day) of rhIL-12 (Genetics Institute, Inc.) by bolus i.v. injection once as an inpatient and then, after a 2-week rest period, once daily for five days every 3 weeks as an outpatient. Therapy was withheld for grade 3 toxicity (grade 4 hyperbilirubinemia or neutropenia), and dose escalation was halted if three of six patients experienced a dose-limiting toxicity (DLT). After establishment of the MTD, eight more patients were enrolled to further assess the safety, pharmacokinetics, and immunobiology of this dose. Forty patients were enrolled, including 20 with renal cancer, 12 with melanoma, and 5 with colon cancer; 25 patients had received prior systemic therapy. Common toxicities included fever/chills, fatigue, nausea, vomiting, and headache. Fever was first observed at the 3 ng/kg dose level, typically occurred 8-12 h after rhIL-12 administration, and was incompletely suppressed with nonsteroidal anti-inflammatory drugs. Routine laboratory changes included anemia, neutropenia, lymphopenia, hyperglycemia, thrombocytopenia, and hypoalbuminemia. DLTs included oral stomatitis and liver function test abnormalities, predominantly elevated transaminases, which occurred in three of four patients at the 1000 ng/kg dose level. The 500 ng/kg dose level was determined to be the MTD. This dose, administered by this schedule, was associated with asymptomatic hepatic function test abnormalities in three patients and an onstudy death due to Clostridia perfringens septicemia but was otherwise well tolerated by the 14 patients treated in the dose escalation and safety phases. The T1/2 elimination of rhIL-12 was calculated to be 5.3-9.6 h. Biological effects included dose-dependent increases in circulating IFN-gamma, which exhibited attenuation with subsequent cycles. Serum neopterin rose in a reproducible fashion regardless of dose or cycle. Tumor necrosis factor alpha was not detected by ELISA. One of 40 patients developed a low titer antibody to rhIL-12. Lymphopenia was observed at all dose levels, with recovery occurring within several days of completing treatment without rebound lymphocytosis. There was one partial response (renal cell cancer) and one transient complete response (melanoma), both in previously untreated patients. Four additional patients received all proposed treatment without disease progression. rhIL-12 administered according to this schedule is biologically and clinically active at doses tolerable by most patients in an outpatient setting. Nonetheless, additional Phase I studies examining different schedules and the mechanisms of the specific DLTs are indicated before proceeding to Phase II testing.

Molecular analysis of melanoma precursor lesions.

Atypical (dysplastic) nevi are melanocytic lesions, which are precursors of melanoma as well as markers of increased melanoma risk. Although these lesions exhibit distinct clinical and histological features, their molecular features are largely unknown. To determine whether atypical, compared to benign nevi, from patients with a clinical history of malignant melanoma reveal molecular changes, we analyzed these lesions for the expression of two growth factors (basic fibroblast growth factor and transforming growth factor alpha), their receptors (fibroblast growth factor receptor-1 and epidermal growth factor receptor), and two cell adhesion molecules (MUC18 and alpha v beta 3 integrin), all of which are expressed in primary and metastatic melanomas. The results demonstrated a statistically significant correlation (P = 0.02) between increasing degrees of histological atypia and expression of epidermal growth factor receptor in the epidermal keratinocytes of atypical melanocytic lesions. Furthermore, both atypical and benign nevi revealed considerably high levels of overall gene activity in their dermal melanocytic and epidermal keratinocytic compartments. In contrast, the epidermal-dermal junction wherein melanoma evolves showed little gene activity, suggesting that molecular events occurring adjacent to this junction may be important for melanocytic transformation.

Postoperative abdominal wall defects with enterocutaneous fistulae.

BACKGROUND: Abdominal wall dehiscence with an associated enterocutaneous fistula is a surgical complication with high morbidity and mortality. Management of the abdominal wall defect is complicated by the continued emergence of liquid bowel contents. PATIENTS AND METHODS: Large abdominal wall wounds of 10 patients with postoperative abdominal wall dehiscence and active enterocutaneous fistulae were managed with early skin grafting directly onto the granulated abdominal viscera. RESULTS: Skin graft take averaged 93 +/- 12%, and there were no perioperative complications related to the skin grafting procedure. Overall mortality was 1 out of 10 patients. Enterocutaneous fistula output did not prove overly injurious to the skin grafts. Wound care was simplified in all but 1 patient with fitting of an ostomy appliance. CONCLUSION: Temporary abdominal wall wound closure with skin grafts improved patient comfort and simplified wound care in a staged reconstructive approach to this surgical complication.

Recurrence rate of diabetic neurotrophic foot ulcers healed using topical application of growth factors released from platelets.

The purpose of this study was to determine the recurrence rate of diabetic neurotrophic foot ulcers that had healed in a treatment protocol using topically applied growth factors released from platelets. Thirty-six patients with diabetic neurotrophic foot ulcers were entered into a randomized prospective double-blind 20-week trial of topically applied platelet growth factors or buffered saline dressings. Ulcers had been present for 15.5 months (mean, range 2 to 60 months). Sixteen patients (44%) healed and were followed up for as many as 30 months to determine the rate of ulcer recurrence. Eleven patients (68.8%) had ulcers that recurred, including 10 treated with platelet growth factors and one treated with buffered saline solution. These ulcers had been present for 20 months (range 5 to 60 months) before healing. Average time to ulcer recurrence was 2.2 months (range 0.25 to 7 months). Five ulcers (31.2%) remained healed, including four treated with platelet growth factors and one treated with buffered saline solution. These healed, nonrecurring ulcers had been present for 5 months (range 2 to 6 months) before healing. Average follow up was 25 months (range 24 to 30 months). Ulcers were more likely to recur if they had been present longer before healing. If patients were not compliant in wearing footwear to protect the healed ulcer, the ulcers were prone to early recurrence. These data indicate that ulcers healed by using growth factors in the form of a platelet growth factors did not have more durable skin over the ulcer and were prone to early ulcer recurrence.

Fascial release as an adjunct to wound closure.

We describe a simple technique to facilitate the primary closure of skin defects. Skin, subcutaneous tissue and fascia are undermined as a unit on both sides of the defect. Fascial releasing incisions are performed, creating two bipedicled fascial flaps attached to the overlying skin. The fascia is approximated, if possible, and the skin sutured. We believe this technique offers a number of advantages and results in a superior closure to that obtained with the conventional method of skin undermining. This approach has enabled the closure of 10 of 11 medium sized wounds.

Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production.

Recent evidence supports the critical and proximate role of IL-12 in regulating both T and NK cell function during inflammation. In these studies, we evaluated the in vivo antitumor activity of murine IL-12 in murine adenocarcinoma and sarcoma models using both systemic and peritumoral administration. Antitumor effects were consistently demonstrated both in models of microdisease, in which IL-12 treatment was initiated soon after tumor inoculation (1 to 5 days), and in animals bearing large established tumors (7 to 14 days). Treatment with IL-12 markedly prolonged survival and, in most cases, caused complete tumor regression. Significant reduction in pulmonary metastases after systemic treatment was observed when treatment was delayed for 10 days after tumor inoculation. Increases in serum IFN-gamma, TNF-alpha, and nitrogen oxides were demonstrated, exceeding those observed with IL-2 treatment. Systemic administration of anti-IFN-gamma Abs before IL-12 treatment nearly completely abrogated the antitumor effect in experiments using subcutaneous tumors or pulmonary metastases. Depletion of the individual T cell subsets CD4 and CD8 by systemic administration of mAbs diminished the effectiveness of IL-12 when administered in combination. An infiltrate composed primarily of CD8+ + cells was demonstrated by using immunohistochemical analysis of tumors after IL-12 treatment. Minimal apparent toxicity was demonstrated at effective doses (0.1 to 1.0 microgram/day) of IL-12. These results indicate that IL-12 is an effective and minimally toxic antitumor agent in murine tumor models and leads to an immune-mediated rejection involving, at least in part, IFN-gamma, CD4+, and CD8+ cells. Human clinical trials of IL-12 for the treatment of malignancy are supported by these studies.